KR20060087693A - Anti-tumor agent containing lrrc3b gene and diagnosis method using the same gene - Google Patents

Abstract

The present invention relates to an anticancer composition comprising a LRRC3B (Leucine rich repeat containing 3B) gene and a method for diagnosing cancer using the gene, in particular an anticancer composition comprising the LRRC3B gene exhibiting excellent cancer formation inhibitory activity and the gene. The present invention relates to a method for diagnosing cancer by measuring abnormal methylation. Anticancer composition and diagnostic method comprising the LRRC3B gene of the present invention can be usefully used for inhibiting and treating cancer.

Methylation, anticancer composition

Description

Anti-cancer composition comprising the LRCR3 gene and diagnostic method using the gene {anti-tumor agent containing LRRC3B gene and diagnosis method using the same gene}             

1A is a diagram illustrating a process of extracting genomic DNA of human cancer cells and performing restriction landmark genomic scanning (RLGS),

N: cleavage site of NotI restriction enzyme

E: Cleavage site of EcoRV restriction enzyme

H: cleavage site of HinfI restriction enzyme

Figure 1b is a diagram showing the results of comparing the RLGS profile of the genomic DNA of 12 gastric cancer cell lines,

Arrow: Spot DNA (3C43)

1C is a graph showing that the concentration of 3C43 spot DNA is significantly decreased in cancer tissues of gastric cancer patients compared to normal tissues.

Figure 2a is a diagram showing the nucleotide sequence determined by sequencing after extracting 3C43 spot DNA of normal tissue,

FIG. 2B is a diagram showing that the nucleotide sequence of FIG. 2A is located at the promoter of the LRRC3B gene and overlaps with the CpG island.

3 is a diagram showing the results of Southern blot analysis using genomic DNA in normal tissue, cancer tissue, lymph nodes, and cell lines.

N: normal tissue T: cancer tissue L: lymph node C: cell line

Figure 4a is a diagram showing the cDNA nucleotide sequence of the human LRRC3B gene,

4B is a diagram showing an amino acid sequence estimated from the cDNA nucleotide sequence of the human LRRC3B gene of FIG. 4A.

FIG. 5 is a diagram showing the results of performing Northern blot on a MTN (multi-tissue nitrocellulose membrane) by labeling the PCR product of FIG. 4A with a radioisotope in order to determine whether LRRC3B gene is expressed in normal human tissues.

6a is a diagram showing the results of RT-PCR separation and total RNA from gastric cancer cell line and breast cancer cell line to analyze the expression pattern of LRRC3B gene,

6B is a diagram showing the results of RT-PCR isolation and total RNA from cancer tissues of gastric and breast cancer patients to analyze the expression patterns of the LRRC3B gene.

FIG. 7 shows the ratio of the expression level of LRRC3B in cancer tissue to the amount of LRRC3B gene expression in normal tissue by performing RT-PCR analysis on total RNA of cancer tissue and normal tissue extracted from gastric cancer patients. According to histopathological classification criteria,

Bar: mean value of expression

8A is a diagram showing a nucleotide sequence in an upper region including an exon 1 site of the human LRRC3B gene,

FIG. 8B is a diagram showing the results of a luciferase assay to verify whether the nucleotide sequence corresponding to the CpG island of FIG. 8A serves as a promoter for the LRRC3B gene. FIG.

Figure 8c shows amplification of genomic DNA of gastric cancer cell line modified with bisulfate using BS primers as a template, cloned into pGemTeasy vector, and sequenced to analyze the presence or absence of methylation at 52 CpG loci. Is shown

Methylated CpG Locus: Black Square

8D is a diagram showing the results of performing MSP using MSP # 1 and MSP # 2 primers as a template of genomic DNA of gastric cancer cell line modified with bisulfite,

M: MSP results for detecting methylated DNA

U: MSP results for detecting unmethylated DNA

Figure 9 shows the total RNA isolated and purified from these cells after treatment with DNA methyltransferase inhibitors or histone deacetylase inhibitors, or both inhibitors, in gastric cancer cell lines and breast cancer cell lines in which LRRC3B is found to be extremely low. Is the result of RT-PCR analysis.

Figure 10 shows that when the expression vector expressing the human LRRC3B gene was prepared and transformed into a gastric cancer cell line in which LRRC3B was expressed very low, the growth rate of the cells was significantly slowed in the experimental group overexpressing the LRRC3B gene compared to the control group. ego,

FIG. 11A is a graph showing the results of in vitro cell growth rate analysis for the SNU601 cell line, which is a low-expression control group of SNU601-LRRC3B # 1, SNU601-LRRC3B # 3, and LRRC3B gene, which stably overexpresses the LRRC3B gene.

11B shows cancer tissues for 57 days after subcutaneous injection of two different SNU601-LRRC3B # 1 and SNU601-LRRC3B # 3, which are stably overexpressing the LRRC3B gene, and the SNU601 cell line, which is a control group with low expression of the LRRC3B gene, in the same amount. The figure shows the result of measuring the change in size.

FIG. 11C is a diagram showing the result of measuring the size of the mouse tissue sacrificed and the cancer tissues extracted on the last day of observation of cancer formation ability in FIG. 11B;

FIG. 11D is a diagram showing a result of directly comparing sizes by sacrificing mice and extracting cancerous tissues on the last day of cancer formation ability observation in FIG. 11C;

FIG. 11E is a diagram showing the results of verifying the activity of the LRRC3B gene using RT-PCR after separating total RNA from three pairs of cancer tissues extracted from the experimental group SNU601-LRRC3B # 1 and the control group SNU601 in FIG. 11D. ,

Figure 12a is to determine the nucleotide sequence of each LRRC3B gene to investigate whether mutation or single nucleotide polymorphism (SNP) occurred in the nucleotide sequence encoding the LRRC3B protein in the genomic DNA of cancer tissue of 80 gastric cancer patients This figure shows the sequencing results of the found SNPs.

FIG. 12B shows A / A and A / T, T / for PCR products of genomic DNA of 80 gastric cancer patients and 21 normal controls using the primers shown in FIG. 4A to retrieve SNPs of LRRC3B gene using PCR This figure shows the result of determining genotype such as T.

The present invention relates to an anticancer composition comprising a LRRC3B (Leucine rich repeat containing 3B) gene and a method for diagnosing cancer using the gene. More specifically, the LRRC3B gene and abnormal methylation of the gene exhibit excellent cancer formation inhibitory activity. The present invention relates to a method for diagnosing cancer by measuring.

Tumors are a group of cells that exhibit excessive proliferation, and are classified into malignant tumors that are difficult to treat and benign tumors that can be easily removed. In particular, cancer is a genetic disease caused by mutations in the oncogene or tumor suppressor gene genes and can be found at the molecular or cellular level. Proteins produced as a result of the expression of cancer genes are networked within cells to regulate signaling for cell division and differentiation. When abnormality occurs in the regulation of the cell differentiation process is blocked and only the division continues to proliferate, the development of an anticancer agent that can reduce the normal signal transmission path is urgently required (Vogelstein and Kinzler, Nat. Med. Vol. 10, 789-799, 2004).

Currently, the three major therapies for cancer are surgical therapy, drug therapy, and radiation therapy. Since pharmacotherapy has less pain associated with treatment and relatively less cancer recurrence after treatment compared to surgical or radiation therapy. Expectations are gathering. Therefore, a number of anticancer drugs have been developed and used to cope with this, and most of these anticancer drugs exhibit anticancer effects by selectively killing actively dividing cells with cancer aberrant growth in mind. These anticancer agents generally have serious problems that can kill long-term use because they have severe side effects such as immune cells and hair follicle cells that are actively dividing cells. Therefore, as the cause of cancer is newly discovered, the development of a new anticancer agent based on it is required.

Looking at the causes of cancer, cancer suppressor genes that block the production of cancer cells by inhibiting abnormal proliferation of cells or by killing specific cells by triggering apoptosis programs due to abnormal activation of cancer-causing genes that cause cancer An abnormality occurs in and occurs. Even if a cell has an abnormally activated cancer gene, once the cancer suppressor gene shows normal activity, the cell does not form cancer and dies. Therefore, in order to become a cancer cell, not only activation of a carcinogenic gene but also an inactivation of a cancer suppressor gene must appear simultaneously in one cell.

One of the mechanisms by which cancer suppressor genes are inactivated is that abnormal DNA methylation occurs in CpG islands in which cytosine and guanine base pairs form islands (Jones and Laird, Nature Genet. Vol. 21, 163-167). , 1999). Methylation of CpG islands is accomplished by DNA methyltransferases, and when methylation accumulates, proteins such as MeCP2 bind to methylated DNA, thereby binding to histone deacetylases. Histone deacetylase removes acetyl groups bound to histones, resulting in a dense DNA structure nearby, which inhibits transcription factor binding and eventually inhibits gene expression (Brown and Strathdee, Trends Mol. Med. Vol. 8 , S43-48, 2002). When gene expression is suppressed by DNA methylation, expression can be restored by using an inhibitor of DNA methyltransferase or an inhibitor of histone deacetylase. Cancer suppressor genes whose expression is suppressed by DNA methylation to date are CDH1, VHL, CDKN2A, CDKL2B, MLH1, DAPK1, GST1, but many cancer suppressor genes are thought to be affected by DNA methylation. .

As described above, DNA methylation and deacetylation play a very important role in gene transcription control of eukaryotic cells, and DNA methyltransferases such as 5-aza-2-deoxycytidine Inhibitors and natural histone deacetylase inhibitors such as trapoxin, trichostatin A, and depudecin are known to inhibit cancer cell proliferation and clinical trials have been conducted to use them as anticancer agents. (Furumai et al., Proc. Natl. Acad. Sci. USA. 98, 87- 92, 2001).

Treatment with anticancer drugs is important, but more importantly, early detection of cancer cells. The reason for the high mortality rate from cancer is that cancer is discovered after metastasis to other organs. Thus, the earlier cancer is detected, the higher the survival rate. For example, in the case of colorectal cancer, the early five-year survival rate of patients reaches 95%, but if metastases to other organs occur, the survival rate drops to around 7% (Etzioni et al., Nat. Rev. Cancer Vol. 3, 243-252, 2003). Therefore, most researchers are working to develop methods and target genes for screening cancer in the early stages. Recently, it has been reported that DNA and RNA derived from cancer cells are detected in body fluids such as plasma or serum of cancer patients, and studies are being actively conducted to isolate these nucleic acids and use them for early diagnosis of cancer. In particular, since DNA derived from cancer cells isolated from body fluids can be amplified by PCR, it is expected to provide a very useful method for early diagnosis of cancer by detecting changes in DNA methylation in cancer cells. Body fluids can also be used as an important tool to determine the prognosis of patients after surgery or to monitor their responsiveness to cancer treatment.

As a result of experiments on DNA obtained from the body fluid of gastric cancer patients, the methylation detection of cancer suppressor genes such as CDH1, CDK2A, CDK2B, DAPK1 and GSTP1 is only 15-58%. Therefore, in order to further increase accuracy, it is necessary to discover new gene targets (Laird, Nat. Rev. Cancer Vol. 3, 253-66, 2003).

Accordingly, the present inventors analyzed cancer-specific methylation of gastric cancer cell lines and gastric cancer tissue genomic DNA by DNA two-dimensional electrophoresis (RLGS), and discovered a new gene, LRRC3B, and the gene product has cancer inhibitory activity. The present invention has been completed by finding that the expression of the LRRC3B gene is reduced due to DNA methylation near LRRC3B exon 1 in various cancer cell lines.

It is an object of the present invention to provide a method for diagnosing cancer by measuring the LRRC3B gene showing an excellent cancer formation inhibitory activity and abnormal methylation of the gene.

In order to achieve the above object, the present invention provides a LRRC3B protein set forth in SEQ ID NO: 2.

The present invention also provides a gene encoding the LRRC3B protein.

The present invention also provides an expression vector comprising the LRRC3B gene.

The present invention also provides a transformed cell line transformed with the expression vector.

The present invention also provides an anticancer composition containing the LRRC3B gene.

The present invention also provides an anticancer composition containing an expression vector containing the LRRC3B gene.

The present invention also provides a composition for anticancer containing a transformed cell line transformed with an expression vector comprising the LRRC3B gene.

The present invention also provides an anticancer composition comprising a substance that controls methylation of a nucleotide sequence including a CpG island near exon 1 of the LRRC3B gene described in SEQ ID NO: 3.

The present invention also provides a method for diagnosing cancer using the LRRC3B gene.

The present invention also provides a microchip for cancer diagnosis comprising an antibody against the LRRC3B protein.

In addition, the present invention provides a method of diagnosing a disease using the SNP of the 919th base of the nucleotide sequence of SEQ ID NO: 1.

Hereinafter, the present invention will be described in detail.

The present invention provides the LRRC3B protein set forth in SEQ ID NO: 2 and the gene set forth in SEQ ID NO: 1 encoding the LRRC3B protein.

We isolated genomic DNA of gastric cancer cell lines and found a 3C43 DNA spot that significantly reduced or lost specific concentration in gastric cancer genome using RLGS method (see FIG. 1 ). 3C43 divides the spot distribution on the RLGS gel into a total of 30 regions and represents the 43rd DNA spot in the 3C region. Analysis of the nucleotide sequence of the 3C43 DNA spot showed that the sequence was 100% identical to the nucleotide sequence located on the chromosome of the human chromosome 3, and the top of the LRRC3B (Leucine rich repeat containing 3B) gene has yet to be identified. 'upstream) and across the transcription initiation region, and also confirmed that the sequences matched the CpG islands (see FIG. 2 ). From the open reading frame appearing between the transcription initiation codon and the transcription termination codon of the LRRC3B gene, 259 polypeptides consisting of the amino acid sequence represented by SEQ ID NO: 2, ie, the LRRC3B protein, are encoded (see FIG. 4B ).

The present invention also provides a composition for anticancer containing the LRRC3B gene.

The LRRC3B gene is a functional gene that is normally expressed in all tissues of the human body such as the prostate, stomach, heart, bladder, small intestine, large intestine, uterus, and skeletal muscle. In cancer cells, exon 1 of LRRC3B gene and CpG island (upstream of cytosine and guanine base pairs) exist in the upstream of the LRRC3B gene, and hypermethylation occurs in this region, which results in LRRC3B gene expression. As a result, LRRC3B gene expression products are lost in cancer cells. This was confirmed by the inventors observing the result of reactivation of the LRRC3B gene as a result of inhibitor treatment that inhibits methylation in cancer cell lines in which the expression of the LRRC3B gene was not detected ( see FIG. 9 ). In addition, LRRC3B gene was introduced into cancer cells and colony forming ability and cell growth rate were observed. As a result, cancer cell growth inhibitory activity was shown ( see FIGS . 10 and 11A ) . 11b to d ) confirmed that the LRRC3B gene shows anticancer activity

The present invention also provides an expression vector containing the LRRC3B gene and an anticancer composition containing the expression vector.

In order to clarify the activity and mechanism of the LRRC3B gene, the present inventors used pDEST12.1-LRRC3B, a LRRC3B expression vector previously prepared in a SNU601 cancer cell line with low expression of the LRRC3B gene, and pDEST12.2, a vector not containing the LRRC3B gene. The colony forming assay was examined by transforming each as a control and culturing the same amount of cells.

As a result, it was found that the growth rate of cancer cells was significantly decreased by about 33% by the expression of LRRC3B gene. From this result, it was confirmed that LRRC3B has a function of inhibiting the growth of cancer cells.

The present invention also provides a transformed cell line transformed with the expression vector and an anticancer composition containing the transformed cell line.

The present inventors produced cell lines SNU601-LRRC3B # 1 and SNU601-LRRC3B # 3 overexpressing the LRRC3B gene transformed with the LRRC3B expression vector pDEST12.1-LRRC3B prepared above.

To analyze the anticancer effect of the transformant, a tumor type performance (tumorigenecity) analysis using a nude mouse was performed. The cancer formation ability analysis was performed after subcutaneous transplantation of three cell lines, such as SNU601, SNU601-LRRC3B # 1, and SNU601-LRRC3B # 3, to the nude mouse subcutaneously to measure tumor size.

As a result, in the case of the control group injected with the SNU601 cell line having a very low expression of the LRRC3B gene, it was observed that cancer was formed rapidly with time. The mice injected with the SNU601-LRRC3B # 1 and SNU601-LRRC3B # 3 of the present invention It was observed that the cancer growth was significantly slowed compared to the control (see FIG. 11A ).

From these results, the present inventors confirmed that the LRRC3B transformant, ie, the LRRC3B gene, has excellent cancer formation inhibitory activity.

The present invention also provides an anticancer composition containing LRRC3B protein as an active ingredient.

The protein which is the product of the LRRC3B gene, ie LRRC3B gene, of the present invention shows excellent anticancer activity. Specifically, it was confirmed that LRRC3B gene expression is inhibited in about 82% of gastric cancer cell lines and all breast cancer cell lines. In addition, inhibition of expression of the LRRC3B gene in various gastric and breast cancer cell lines is due to abnormal DNA methylation in typical CpG islands located near exon 1 of the LRRC3B gene, by treatment with DNA methyltransferase inhibitors or histone deacetylase inhibitors. It was confirmed that expression of the LRRC3B gene can be induced. Therefore, the LRRC3B gene and its gene products of the present invention can be usefully used not only for the development of anticancer drugs but also for the development of a test method for diagnosing cancer by measuring abnormal DNA methylation near the exon 1 of the LRRC3B gene. In addition, it can be used for the development of an active substance that induces the expression of the LRRC3B gene for the treatment of cancer in which the expression of LRRC3B is suppressed. Accordingly, the present invention provides an anticancer composition comprising a substance that modulates methylation of a nucleotide sequence including a CpG island (see FIG. 8A ) in the vicinity of exon 1 of the LRRC3B gene described in SEQ ID NO: 3.

In addition, the present invention

1) separating and purifying genomic DNA from the subject's tissue;

2) modifying the genomic DNA of step 1;

3) amplifying a portion of the nucleotide sequence of the CpG island of LRRC3B in the genomic DNA of step 2 by methylation-sensitive PCR (MSP);

4) It provides a cancer diagnostic method comprising the step of detecting the methylation of the base sequence containing the CpG island near exon 1 of the LRRC3B gene from the product of step 3.

The inventors treated sodium hydroxide (NaOH), hydroquinone and sodium bisulfite for the DNA modification of step 2).

In step 2), the MSP method uses the principle that C (cytosine) of methylated CpG remains unmodified when DNA is modified, but C of CpG is converted to T (thymine) in the case of non-methyl C. PCR is performed using two kinds of primers designed as C (PCR-M) or T (PCR-U). If the target gene region is methylated, the PCR product appears in the PCR-M primer. U uses the principle that no PCR product appears.

In addition, the present invention

1) separating and purifying the genomic DNA from the cancer tissue of the subject;

2) cleaving the genomic DNA of step 1 with a restriction enzyme that is sensitive to methylation of DNA and a restriction enzyme that is not sensitive to methylation;

3) performing PCR using a portion of a nucleotide sequence including a CpG island near exon 1 of the LRRC3B gene as a template using the DNA of step 2 as a primer;

4) It provides a cancer diagnostic method comprising the step of detecting the methylation of the base sequence including the CpG island near exon 1 of the LRRC3B gene described in SEQ ID NO: 3 from the difference in the reaction product of step 3.

Restriction enzymes sensitive to DNA methylation in step 2) can cleave only unmethylated DNA, not methylated DNA. On the other hand, enzymes that are not sensitive to DNA methylation cleave both methylated and unmethylated DNA, so when the same DNA is treated with the two restriction enzymes, the DNA sites with different fragment sizes are methylated sites.

In addition, the present invention

1) separating and purifying genomic DNA from the subject's tissue;

2) cleaving the genomic DNA of step 1 with a restriction enzyme sensitive to DNA methylation;

3) electrophoresis of the genomic DNA of step 2 followed by transfer to the membrane;

4) hybridizing the membrane of step 3 with a DNA probe labeled with radioisotopes on the CpG sequence of the LRRC3B gene;

5) providing a method for diagnosing cancer comprising exposing the membrane of step 4 to an X-ray film to detect methylation of a nucleotide sequence comprising a CpG island near exon 1 of the LRRC3B gene.

We used a highbond-N + nylon membrane as the membrane for transferring genomic DNA in step 2 and the radioisotope was prepared by labeling with [α- ³² P] dCTP.

In addition, the present invention

1) separating and purifying total RNA from the subject's tissue;

2) amplifying the expressed mRNA by RT-PCR using primers prepared with the entire RNA of step 1 as a partial sequence of the LRRC3B gene;

3) It provides a cancer diagnostic method comprising the step of detecting the expression of the LRRC3B gene from the amplified RNA of step 2.

In addition, the present invention

1) separating and purifying total RNA from the subject's tissue;

2) transferring the entire RNA of step 1 to the membrane after electrophoresis;

3) hybridizing the LRRC3B gene labeled with the radioisotope to the membrane of step 2 with an RNA probe;

4) It provides a cancer diagnostic method comprising the step of detecting the expression level of the LRRC3B gene by exposing the membrane of step 3 to an X-ray film.

The present invention also provides a microchip for cancer diagnosis comprising an antibody against the LRRC3B protein.

Microchips are manufactured by combining molecular biology knowledge with electrotechnical technology. At the same time, at least hundreds of genes or proteins can be searched in a short time, making new gene search and diagnosis quick and easy. Therefore, a microchip using an antibody to a part of the LRRC3B cDNA sequence or an LRRC3B protein produced therefrom can be used to diagnose a large number of patients suspected of various cancers including gastric cancer and breast cancer. In the present invention, especially in the stomach cancer (EGC, early gastric cancer (EGC) and advanced gastric cancer (AGC, advanced gastric cancer) between the two groups, such as the average expression level of the LRRC3B gene in early gastric cancer was found to be significantly lower (See FIG. 7 ) The loss of function of LRRC3B gene in the development of gastric cancer is expected to be effectively used for early diagnosis of cancer.

In addition, the present invention provides a method of diagnosing a disease using the SNP of the 919th base of the nucleotide sequence of SEQ ID NO: 1.

The inventors have discovered a new single nucleotide polymorphism (SNP) that has not been reported so far in the sequencing process (see FIG. 12A ). The new SNP is the 919th base of the nucleotide sequence shown in SEQ ID NO: 1, and the amino acid substitution is performed by replacing T with the T, the third nucleotide of GGA designating glycine (gly), the 110th amino acid of FIG. 4B. It is a base change that does not affect. In order to verify the SNP in a more economical and faster way than sequencing, the forward primer LRRC3B-919-forward and two reverse primers LRRC3B-919A-reverse and LRRC3B-919T-reverse of the present invention can be used. (See FIG. 4A ).

The newly discovered SNPs in the present inventors have no significance associated with the development of gastric cancer, but are valuable as a useful marker for the composition of haplotype maps using SNPs of the entire genome of humans and for their association with specific diseases. high.

  Hereinafter, the present invention will be described in detail by way of examples.

only. The following examples are merely illustrative of the present invention, but the content of the present invention is not limited to the following examples.

Example 1 LRRC3B gene discovery

The present inventors searched for abnormal methylation specifically found in cancer cell genes in order to discover genes related to cancer in gastric cancer cell lines and gastric cancer tissues, and found CpG islands, which are hypermethylated sites of the LRRC3B gene.

<1-1> Restriction landmark genomic scanning (RLGS) analysis

First, we isolated genomic DNA from gastric cancer tissue and normal tissue extracted from 11 gastric cancer cell lines and 15 patients. Purification of genomic DNA was performed by freezing and grinding the tissue in liquid nitrogen following the procedure of Samblook and Russell (Molecular Cloning, CSHL Press, 3rd ed. 2001), suspended in 10 ml of RNase-containing Proteinase buffer and 20 at 37C. After the reaction, Proteinase K was added at a final concentration of 1 mg / ml and the reaction was carried out at 65C for 1 hour. After the reaction, the solution was treated with phenol / chloroform and ethanol, and then spulled out to recover only the final polymer DNA. All purified genomic DNAs were treated with restriction enzymes and subjected to two-dimensional electrophoresis to confirm the size of the fragments. Restriction landmark genomic scanning (RLGS) ( FIG. 1A ) was used for cell lines versus normal tissues and normal tissues. Scanned profiles of cancerous tissues were compared. Electrophoresis of genomic DNA with restriction enzymes resulted in over 2,000 spot DNAs, which are enzyme cut fragments, on gels. Of the 3C43 DNA spots, 10 cancer cell lines out of 12 cancer cell lines Spot loss was observed at (83%) ( FIG. 1B ) and spot DNA concentration was reduced (80%) in cancer tissues of 12 of 15 patients ( FIG. 1C ).

<1-2> Hypermethylated 3C43 Spot DNA Isolation and Cloning

3C43 spot DNA was recovered from electrophoresis gels subjected to RLGS according to the method of Nagai et al. (Nagai et al., BBRC, 213: 258-265, 1995). First, the 3C43 region was cut out from the electrophoretic gel, dissolved in DNA recovery solution (66 mM Tris, 6.6 mM MgCl ₂ , 10 mM DTT, 150 mM NaCl, 10% PEG6000), and then the supernatant was obtained by centrifugation, and the ethanol was precipitated. DNA was recovered. This was again dissolved in the recovery solution and conjugated with a NotI and HinfI linker with a ligase (T4 ligase), and PCR reaction was performed using the linker sequence. PCR reaction conditions, using the DNA polymerase (Taq polymerase, Takara) denatured primer pair and template described above for 2 minutes at 94 ℃, 30 seconds at 94 ℃, 30 seconds at 55 ℃ and 72 ℃ The reaction was completed 35 times for 1 minute at, and extended for 5 minutes at 72 ° C. to terminate the reaction. This amplified 3C43 spot DNA and cloned it into pBlue-KS (+) vector and named it pBlue-KS (+) 3C43.

<1-3> High methylation 3C43 spot DNA sequencing and LRRC3B discovery

The nucleotide sequence of pBlue-KS (+) 3C43 cloned above was analyzed ( FIG. 2A ) and the nucleotide sequence of the LRRC3B gene located in the human chromosome 3 armband was examined using the Blot program in the UCSC genome database. It was confirmed that it is 100% identical to Exon 1 and CpG islands upstream of the gene ( FIG. 2B ).

<1-4> Promoter Activity Analysis of CpG Island of LRRC3B Gene

The inventors examined whether CpG islands act as promoters of the LRRC3B gene, just as exon 1 of the LRRC3B gene and CpG islands upstream of the genes are present as other promoters upstream of the genes.

Specifically, the 696bp nucleotide sequence of DNA described in SEQ ID NO: 1 as a template and using primers described in SEQ ID NO: 4 and 5 corresponding to the CpG island of LRRC3B was amplified by PCR (Fig. 8a). PCR reaction conditions, using the DNA polymerase (Taq polymerase, Takara) denatured primer pair and template described above for 2 minutes at 94 ℃, 40 seconds at 94 ℃, 50 seconds and 72 ℃ The reaction was completed 35 times for 1 minute at, and extended for 5 minutes at 72 ° C. to terminate the reaction. This pGL3-LRRC3BCpG was prepared by cloning into a pGemT easy vector (Promega) and double cutting DNA fragments with restriction enzymes NcoI and SacI into pGL3-Basic vector (Promega) containing a luciferase gene. 1 μg of each of the control group pGL3-Basic DNA and the experimental group pGL3-LRRC3BCpG DNA were transformed into a Hela cell line using lipofectamine (Invitrogen) and analyzed for luciferase activity.

As a result, pGL3-basic-S03CpG71 luciferase activity including the nucleotide sequence of CpG island was significantly higher than that of the control group pGL3-basic 48 hours after transformation, so that the promoter activity of pGL3-basic-S03CpG71 was higher than that of pGL3-basic. It is conceivable ( FIG. 8B ) which indicates that the region of the CpG island acts as a promoter of the LRRC3B gene.

<1-5> Southern blot analysis

Southern blot analysis was performed to determine whether the DNA sequence obtained in Example <1-3> was lost or decreased in cancer cells due to hypermethylation or chromosomal deletion. 10 μg of genomic DNA of normal tissue, cancer tissue, lymph node tissue, and cancer cell lines of patients whose cancer cells have spread to lymph nodes were cut or double-cut with restriction enzymes such as NotI, EcoRV, NotI and HinfI, respectively, and then subjected to 1.2% agarose gel. Electrophoresis and transfer to high bond-N + nylon membrane. The DNA probe was used by labeling the pBlue-KS (+) 3 C43 DNA cloned in Example <1-2> with [α- ³² P] dCTP (3,000 Ci / mmol, NEN). The hybridization reaction was performed on a high bond-N + nylon membrane twice with 15 × CPE / 0.1% SDS in a 5 × SSPE / 0.1% SDS solution containing 10 ⁶ cpm / ml of probe DNA and 15 min at 60 ° C with 0.1 × SSPE / 0.1% SDS solution. Washed twice. The reaction results were detected by developing the membrane after exposure to Kodak XAR-5 film at −70 ° C. for 16 hours ( FIG. 3 ).

As a result, in the case of HinfI / HinfI cleavage, which cleaves DNA even when methylation occurs at methylation-insensitive restriction enzyme, that is, restriction enzyme recognition site, distinct DNA bands are observed in all genomic DNA such as normal tissue, cancer tissue, lymph node tissue and cell line. When NotI, a methylation-sensitive restriction enzyme, was used, the size of DNA fragments changed as NotI could not cut methylated restriction enzyme sites in genomic DNA of cancer tissues and lymph nodes and cell lines compared to normal tissues. It was confirmed ( FIG. 3 ).

Therefore, the loss or concentration of 3C43 spot DNA by RLGS analysis in gastric cancer cell line and gastric cancer tissue is not caused by chromosomal deletion or loss of heterozygosity (LOH), but methylation of NotI restriction enzyme recognition site of 3C43 DNA fragment. Proved to be the main cause.

<Example 2> Analysis of LRRC3B gene expression in normal tissues, cancer cell lines and cancer tissues

The present inventors investigated the expression patterns of various LRRC3B genes including a part of the methylated 3C43 DNA found in Example 1 , and analyzed abnormal expression of LRRC3B genes in cancer cell lines and cancer tissues.

<2-1> Northern Blot Analysis

The inventors performed Northern blot analysis with 'Human Muscle MTN Blot' (Cat # 7765-1) provided by Clontech to determine whether the LRRC3B gene is normally expressed in various human tissues. DNA probes were SEQ ID NO: 6 and [α- ³² P] of the PCR product obtained by using the RT-PCR primer is a forward primer and a reverse LRRC3B LRRC3B primer (Fig. 4a) for being written in 7 dCTP (3,000 Ci / mmol, NEN 4) was prepared by labeling. Hybridization reactions were performed using a high-bond-N + nylon membrane twice with 15 × CPE / 0.1% SDS in a 5 × SSPE / 0.1% SDS solution containing 10 ⁶ cpm / ml of probe DNA and 15 minutes at 65 ° C. with a 0.1 × SSPE / 0.1% SDS solution. It was performed by washing twice. The membrane was exposed to Kodak XAR-5 film for 16 hours at −70 ° C. and then developed to detect reaction results ( FIG. 5 ).

As a result, there was a difference in expression levels depending on the tissue, but distinct LRRC3B mRNA bands were detected in tissues such as the prostate, stomach, heart, bladder, small intestine, large intestine, uterus, and skeletal muscle. Detected. Therefore, it was confirmed that the LRRC3B gene is a functional gene normally expressed in all tissues of the human body.

<2-2> RT-PCR Analysis of Gastric and Breast Cancer Cell Lines

In order to investigate whether the LRRC3B gene found by the present inventors has decreased expression in cancer cell lines unlike normal cells, mRNA of LRRC3B gene was expressed by RT-PCR method after separating total RNA from 11 gastric cancer cell lines and 6 breast cancer cell lines. It was investigated.

SNU1, SNU5, SNU16, SNU216, SNU484, SNU520, SNU601, SNU620, SNU638, SNU668, SNU719 were used as gastric cancer cell lines, and MCF-7, ZR-75-1, Hs578T, MDA-MB- were used as breast cancer cell lines. 231, SCC-1395, SK-BR-3 and the like were used. The cell lines were used by the Korea University Cell Bank (KCLB: http://cellbank.snu.ac.kr/index.htm ), Seoul National University Hospital.

Total RNA was isolated and purified from each cancer cell line using the RNeasy kit (Qiagen) according to the instructions given. Purified RNA is treated with DNAase (DNase I, Gibco-BRL) to remove DNA, and the oligo-dT (Oligo-dT) primer (Gibco-BRL), SuperScript II RNase H ^- kit (Invitrogen) CDNA was synthesized. On the basis of the cDNA, LRRC3B mRNA expression was analyzed by RT-PCR method using forward LRRC3B primers and reverse LRRC3B primers as shown in SEQ ID NOs: 6 and 7. For RT-PCR of control experiments, H-actin forward primers as set out in SEQ ID NO: 8 and H-actin reverse primers as set out in SEQ ID NO: 9 were prepared using β-actin cDNA sequences in humans.

RT-PCR reaction conditions were pre-treated at 94 ° C. for 2 minutes using a gene amplification device (Perkin-Elmer Cetus, model 9600), followed by denaturation at 94 ° C. for 40 seconds, and annealing at 60 ° C. for 50 seconds. Step, the enzyme step (extension) at 72 ℃ 1 was repeated 30 times in succession. After completion of the reaction, all of the reaction solution was electrophoresed on 2% agarose gel and observed by staining with ethidium bromide.

RT-PCR response to β-actin gene for control experiments was pretreated at 94 ° C for 2 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 68 ° C for 1 minute, and 72 ° C for 1 minute. The extension step was repeated 20 times in succession. After completion of the reaction, 3 μl of the reaction solution was electrophoresed on a 2% agarose gel and observed by staining with ethidium bromide.

As a result, RT-PCR product of 516 bp, which is the expected size of LRRC3B gene, was observed in most gastric cancer cell lines, but expression of LRRC3B gene was very low except for SNU484 and SNU520. In addition, as a result of analyzing the expression of the LRRC3B gene in six breast cancer cell lines in the same manner, it was confirmed that the expression of the LRRC3B gene is very low in all cell lines ( FIG. 6A ).

<2-3> RT-PCR Analysis of Gastric and Breast Cancer Tissues

In the same manner as in Example <2-2> , total RNA was isolated from cancer tissues and normal tissues extracted from 269 gastric cancer and 18 breast cancer patients, and the expression of LRRC3B gene was examined using RT-PCR method.

As a result, if the ratio of LRRC3B expression in the cancer tissue of the same patient to the LRRC3B expression in the normal tissue of one patient was defined as loss of gene expression. At this time, the expression loss of LRRC3B gene was observed in 30% of the 269 gastric cancer patients ( Fig. 6b ). In addition, gene expression loss was observed in the tissues of 18 breast cancer patients in all cancer tissues ( FIG. 6B ). Therefore, the above results strongly suggest that the loss of LRRC3B function due to the loss of the LRRC3B gene in gastric cancer tissue and breast cancer tissue is a major cause of gastric cancer and breast cancer.

<2-4> Correlation Analysis of Gastric Cancer Progression and Loss of LRRC3B Gene Expression

The present inventors compared and analyzed the amount of LRRC3B gene expression in normal and cancerous tissues to investigate the association between the transition from early gastric cancer to advanced gastric cancer and loss of LRRC3B gene expression.

As a result, there was no significant difference in the mean expression level of the LRRC3B gene in the intestinal type (I, intestinal type) and diffuse type (D, diffuse type) according to Lauren's classification of gastric cancer (P). = 0.8447). However, in the early gastric cancer (EGC) and advanced gastric cancer (AGC), the T test showed a very low expression level of the LRRC3B gene in early gastric cancer (P, 0.0001). In IA stage, the average expression level of LRRC3B gene was much lower than other stages (P = 0.0002) ( FIG. 7 ). For reference, in the TNM stage, T is a classification system according to tumor invasion, and divided into Tx->T0->T1->T2->T3-> T4 according to the progression level, and N represents lymph node metastasis. According to the number, Nx->N0->N1->N2-> N3. M represents the degree of metastasis to other organs and is classified as Mx->M0-> M1. The above three parameters are used to classify cancer progression or stage of cancer, which is called TNM stage, and from the initial stage, stage is classified into IA->IB->II->IIIA->IIIB-> IV. Therefore, IA is a good prognosis for early gastric cancer, and the prognosis is worse as the disease goes backward.

Therefore, the above results indicate that the loss of LRRC3B gene function due to the loss of LRRC3B gene expression in the early stage of gastric cancer development, and the loss of the function of the LRRC3B gene is likely to occur from the intestinal metaplasia. Loss of function can be presumed to be related to methylation at the CpG island of LRRC3B as shown in the results of Example <1-1> .

<Example 3> Association analysis between LRRC3B gene expression and CpG island methylation

In this regard, the present inventors compared the expression of LRRC3B gene and methylation in CpG island near exon 1 of LRRC3B gene in order to determine whether the expression loss of LRRC3B gene is due to DNA methylation in CpG island present in the gene. It was.

<3-1> Association analysis between LRRC3B gene expression and methylation of CpG islands in cancer cell lines

To investigate that methylation in the CpG island of the LRRC3B gene is a direct cause of loss of expression of the LRRC3B gene, the methylation status for the CpG loci was analyzed in gastric cancer cell lines.

First, 1 μg of each genomic DNA of 11 gastric cancer cell lines was denatured with 0.3 M sodium hydroxide (NaOH) and treated with hydroquinone and sodium bisulfite to obtain modified DNA. Each PCR product was obtained using a BS forward primer described in SEQ ID NO: 10 as a template and a BS reverse primer described in SEQ ID NO: 11 ( FIG. 8A) . The PCR product was cloned into pGEMT easy vector (Promega), and the base sequence of pGEMT-CpG of 6 clones per cell line was determined to analyze the presence or absence of methylation of 57 CpG loci ( FIG. 8C ).

As a result, CpG islands were severely methylated over 44-95% of 10 cell lines except SNU484 (SNU001, SNU005, SNU016, SNU216, SNU520, SNU601, SNU620, SNU638, SNU668, SNU719). In the remaining 9 cell lines except SNU520, it was confirmed that the expression of the LRRC3B gene is very low as shown in Figure 6a , and in the SNU484 LRRC3B gene was normally expressed and the degree of methylation was 2%. The results can confirm that hypermethylation of the LRRC3B gene on the CpG island is a direct cause of the loss of gene expression of LRRC3B.

<3-2> Analysis of LRRC3B CpG Island Methylation Status by Methylation-sensitive PCR

In addition to sequencing, the present inventors introduced the MSP method to enable a simple and fast analysis of the methylation status of the CpG islands of the LRRC3B gene. When MSP transformed DNA to sodium bisulfite as in Example <3-1> , C (cytosine) of methylated CpG remains unmodified but C of CpG for unmethylated C Is used to transform T into thymine. With two types of primers designed as C (PCR-M primers) or T (PCR-U primers) for the same loci of CpG islands (the cytosine and guanine base pairs interspersed in island shapes) When PCR is performed, if the desired gene region is methylated, the PCR product appears in the PCR-M primer, and if it is not methylated, the PCR product appears in the PCR-U.

Sodium bisulfite is treated in the above using modified DNA MSP was performed using two sets of primers, MSP # 1 and MSP # 2 ( FIG. 8A ). As a result, when MSP # 1 was used as shown in FIG. 8D , PCR-M was used in 7 cell lines (SNU001, SNU005, SNU601, SNU620, SNU638, SNU668, and SNU719) among 9 cell lines with very low expression of LRRC3B. PCR products were generated by the primers, and the product was observed in U (non-methyl DNA) in two cell lines (SNU016 and SNU216) that exhibited relatively low methylation compared to other cell lines. Thus, the results indicate that the position of the primer of MSP # 1 is a region methylated in 7 cells but not methylated in both cell lines.

Therefore, we performed MSP by selecting a region where MSP shows a direct correlation with LRRC3B gene expression. As a result, when MSP # 2 was used, products were observed in M and U in both cell lines SNU016 and SNU216. Therefore, it can be seen that the reaction conditions of MSP # 2 are consistent with those of FIG. 8C . These results indicate that the primers of MSP # 2 can more accurately analyze the methylation pattern of LRRC3B CpG island. It was confirmed that it can be usefully used for development.

<3-4> Reactivation of LRRC3B Gene

In order to analyze whether the DNA methylation observed in Example <3-2> was a direct cause of inhibiting the expression of the LRRC3B gene, the researchers expressed the expression of the LRRC3B gene when the DNA methyltransferase inhibitor or histone deacetylase inhibitor was treated. It was investigated if it was reactivated.

Specifically, DNA methyltransferase inhibitors were selected by randomly selecting two gastric cancer cell lines SNU216 and SNU601 and two breast cancer cell lines Hs578T and SK-BR-3 among cell lines for which the expression of the LRRC3B gene was not detected by RT-PCR analysis. Phosphorus 5-aza-2-deoxycytidine (AZA, 300 nM) or histone deacetylase inhibitor trichostadin A (TSA, 1 nM) alone or two inhibitors Were treated simultaneously. 1 day or 3 days post-treatment, total RNA isolated according to the same manner as in Example <2-2> from each cell line was purified and SEQ ID NO: 6 and 7 The expression of the LRRC3B gene was analyzed by RT-PCR method using primer pairs as described below. The reaction conditions were pre-treated at 94 ° C. for 2 minutes, followed by denaturation at 94 ° C. for 40 seconds, annealing at 60 ° C. for 50 seconds, and repeated enzymatic reaction at 72 ° C. for 1 minute. It was.

As a result, it was confirmed that the expression of the LRRC3B gene was induced again when the DNA methyltransferase inhibitor or histone deacetylase inhibitor was treated (FIG. 9). Inhibitor treatment that inhibited methylation resulted in reactivation of the LRRC3B gene and demonstrated that the expression of the LRRC3B gene was suppressed by hypermethylation of CpG islands present near exon 1 of the LRRC3B gene in gastric and breast cancer cell lines.

Example 4 Preparation of SNU601-LRRC3B # 1 and SNU601-LRRC3B # 3 Cell Lines

The present inventors produced a cell line overexpressing the LRRC3B gene in order to elucidate the activity and mechanism of action of the LRRC3B gene as follows.

<4-1> Preparation of LRRC3B Gene Expression Plasmid Vector

Specifically, the plasmid vector expressing the LRRC3B gene is a 780 bp DNA fragment containing the entire coding region in the cDNA of the LRRC3B gene using the LRRC3B gene represented by SEQ ID NO: 1 as a template and a forward primer LRRC3B and a reverse primer LRRC3B. 4A ) was obtained. The reaction conditions were pre-treated at 94 ° C. for 2 minutes, followed by denaturation at 94 ° C. for 40 seconds, annealing at 60 ° C. for 50 seconds, and repeated enzymatic reaction at 72 ° C. for 1 minute. It was. It was prepared by inserting the pDEST12.1 vector in the forward direction and named pDEST12.1-LRRC3B.

<4-2> Colony forming assay

The present inventors transformed the above-prepared LRRC3B expression vector pDEST12.1-LRRC3B and control vector pDEST12.1 into SNU601 cancer cell line with low expression of LRRC3B gene to reveal the activity and mechanism of LRRC3B gene in cancer cells. And colony forming assay (Colony forming assay) was investigated by incubating the transformed cancer cell lines (1 x 10 ⁴ cells / well) in soft agar.

As a result of culturing the cells for 3 days independently and repeating for 20 days, the control group transformed with pDEST12.1 formed 113 ± 14 colonies (100-129) per plate, whereas the pDEST12.1-LRRC3B was transformed. In the experimental group, 37 ± 5 colonies (31 to 41) were formed on average ( FIG. 10 ). Therefore, it was found that the growth rate of cancer cells was significantly decreased by about 33% by the expression of LRRC3B gene. From this, it was confirmed that LRRC3B has a function of inhibiting the growth of cancer cells.

<4-3> Stable transfection

We prepared the LRRC3B gene by using the plasmid vector pDEST12.1-LRRC3B and neomycin selection marker (pDEST12.1 plasmid) prepared from Example 4 above to produce a cell line overexpressing the LRRC3B gene. After co-transfection with lipofectamine (Lipopectamin, Gibco-BLR) in a non-present SNU601 cell line, a stable transfectant in which pDEST12.1-LRRC3B was stably introduced was G418. Were selected. The cell lines overexpressing the LRRC3B gene thus selected were named SNU601-LRRC3B # 1 and SNU601-LRRC3B # 3, respectively.

Example 5 Investigation of Tumorogenicity of Tumorogenicity of LRRC3B Gene

The inventors hypothesized that the LRRC3B gene will have an anticancer effect, as observed in the results of the colony forming ability analysis of Example <4-2> , and the cell growth rate in vitro and tumorigenicity using nude mice. ) Analysis was performed.

<5-1> In vitro cell growth rate measurement

First, in vitro cell growth rates of the overexpressed cell lines SNU601-LRRC3B # 1 and SNU601-LRRC3B # 3 of the LRRC3B gene established in Example <4-3> were analyzed.

Cells were inoculated at 5 x 10 ⁴ cells / well in “Day 0” and measured for 24 hours. To measure the cell volume, 50% TCA (trichloroacetic acid) was added to the plate and left at 4 ° C. for 60 minutes to fix, wash, and dry the cells. Stained with 0.4% SRB (sulforhodamine B) solution for 30 minutes, washed with 0.1% acetic acid and dried, and then dissolved in 100 μl of 10mM Tris Base (pH 10.5) to measure absorbance at 540 nm. In this same manner, the cell growth changes were confirmed by measuring the amount of cells on the second, third and fourth days.

As a result, the SNU601-LRRC3B # 1 and SNU601-LRRC3B # 3 cells had a decrease of 16.5% and 34.4% on day 2 and 50.8% and 4.4% on day 3, respectively, compared to the control untransformed SNU601 cell line. A decrease in cell growth rate was observed. On day 4, the last day, cell growth rates of 137.6% (SNU601-LRRC3B # 1) and 57.4% (SNU601-LRRC3B # 3) were observed ( FIG. 11A ).

<5-2> Investigation of Tumorogenicity Inhibitory Activity by Nude Mouse Transplantation Test

The present inventors performed cancer tumorigenicity analysis using nude mice to analyze the anticancer effect of the LRRC3B gene.

Specifically, each cell line of untransformed SNU601, SNU601-LRRC3B # 1, and SNU601-LRRC3B # 3, which were the control groups, was subcutaneously transplanted in the same amount (3 × 10 ⁷ cells / ml) in nude mice by 0.3 ml. After the subcutaneous injection, the tumor size was measured from the 13th day until the final day, 57 days. The tumor size was calculated by measuring the three directions using a vernier caliper. On day 57, the last day, nude mice were sacrificed to excise tumors and weigh.

As a result, in the control group injected with methylated SNU601 cell line, it was observed that cancer was formed rapidly over time, and the mice injected with SNU601-LRRC3B # 1 and SNU601-LRRC3B # 3 showed significantly higher cancer growth than the control group. Slowing was observed ( FIG. 11B ).

Specifically, the cancer tissue size of the transplant group such as SNU601-LRRC3B # 1 and SNU601-LRRC3B # 3 on the last day (day 57) were 108.0 ± 53.2 mm 3 and 0.5 ± 1.1 mm 3, respectively, compared to 480.6 ± 117.0 mm 3 of the control group SNU601, respectively. Tumor size reductions of 77.5% and 99.9% were observed (p <0.001, FIG. 11C ). On the same day, the cancer tissues were excised and weighed. 370.3 ± 244.4 mg and 5.2 ± 6.6 mg of SNU601-LRRC3B # 1 and SNU601-LRRC3B # 3, respectively, compared to 1114.0 ± 342.9 mg of SNU601, the control group. Tumor weight was decreased by 66.8% and 99.5%, respectively (p <0.001, FIG. 11D ).

<5-3> RT-PCR Analysis of Cancer by Nude Mouse Transplantation Test

In order to confirm whether the LRRC3B gene activity is maintained in cancer tissues obtained from nude mice in addition to the cancer formation inhibitory activity observed in the nude mouse transplantation experiment, the present inventors 3 extracted from mice transplanted with SNU601 and SNU601-LRRC3B # 1. Each total RNA was isolated and purified from dog cancer tissues using the RNeasy kit (Qiagen) according to the instructions presented. Purified RNA is treated with DNAase (DNase I, Gibco-BRL) to remove DNA, and the oligo-dT (Oligo-dT) primer (Gibco-BRL), SuperScript II RNase H ^- kit (Invitrogen) CDNA was synthesized. RT-PCR was performed using the primers set forth in SEQ ID NOs: 6 and 7 to investigate the activity of the LRRC3B gene. The reaction conditions were pre-treated at 94 ° C. for 2 minutes, followed by denaturation at 94 ° C. for 40 seconds, annealing at 60 ° C. for 50 seconds, and repeated enzymatic reaction at 72 ° C. for 1 minute. It was.

As a result, it was observed that the cancer tissues extracted from SNU601 transplanted mice had very low activity of the LRRC3B gene, but the cancer tissues extracted from SNU601-LRRC3B # 1 transplanted mice were overexpressed in the LRRC3B gene. Based on the above results, the present inventors observed that LRRC3B maintains gene activity in cancer tissues and confirmed that LRRC3B gene has cancer formation inhibitory activity (FIG. 11D) .

Example 6 Mutation and Monobasic Polymorphism (SNP) Analysis of LRRC3B Gene

We investigated mutations or single nucleotide polymorphisms (SNPs) in the LRRC3B gene to investigate whether decreased or lost expression of the LRRC3B gene in cancer cells involves other genetic mechanisms besides methylation in CpG islands. It was.

<6-1> Analysis of the nucleotide sequence of the protein coding region of the LRRC3B gene

First, the inventors confirmed sequencing of 780 bp of the LRRC3B gene nucleotide sequence including the entire coding region of the LRRC3B gene described in SEQ ID NO: 1 in cancer tissues of 80 patients. As a result, no mutation was observed in the nucleotide sequence encoding the protein of LRRC3B in the cancer tissue of the patient, from which the inventors found that the decrease in the expression of the LRRC3B gene observed in cancer tissue of gastric cancer patients is not due to the mutation. Confirmed.

However, the inventors have discovered a new single base polymorphism (SNP) that has not been reported so far in the sequence analysis ( Fig. 12a ). This new SNP is the 919th base of FIG . 4A , the base of GGA designating glycine (gly), the 110th amino acid of FIG. 4B , where A is the third nucleotide of GGA, and has no effect on amino acid substitution. It is a change. We observed A / A homozygotes, A / T heterozygotes, and T / T homozygotes in 53, 23, and 4 patients, respectively, in cancer tissues of 80 patients with gastric cancer. The frequency of A and T alleles was analyzed as 80.6% (129/160) and 19.4% (31/160), respectively.

<6-2> Determination of SNP Type in LRRC3B Gene by PCR

The inventors performed PCR to verify the above SNP results. For this purpose, a forward primer LRRC3B-919-forward as set forth in SEQ ID NO: 20 and two reverse primers LRRC3B-919A-reverse and LRRC3B-919T-reverse as described in 21 and 22 were designed ( FIG. 4A ), using 80 PCR was performed on normal tissue DNA of gastric cancer patients and blood DNA of 21 normal healthy patients without gastric cancer history. The reaction conditions were pretreatment at 94 ° C. for 5 minutes, followed by denaturation at 94 ° C. for 45 seconds, annealing at 60 ° C. for 35 seconds, and extension at 72 ° C. for 2 minutes. Repeated 35 times.

As a result, in the case of gastric cancer patients, it was confirmed that the SNP can be accurately determined using PCR in the same manner as the result of sequencing. In normal subjects, A / A homozygotes, A / T heterozygotes, and T / T homozygotes were observed in 11, 9, and 1 patients, respectively, and thus, A and T alleles. The incidence of 73.8% (31/42) and 26.2% (11/42), respectively, and the chi-square test of the distribution of specific genotypes and alleles between gastric cancer patients and normal subjects showed significant differences between the two groups. Did not appear.

However, the SNP newly discovered by the present invention is considered to be highly useful as a marker useful for constructing a haplotype map using SNPs of the entire genome of humans and analyzing the association with specific diseases.

As described above, the LRRC3B gene found by the present inventors is a functional gene normally expressed in various human tissues. The loss of function due to abnormal DNA methylation in the CpG island of the gene is one of the main causes of carcinogenesis. Therefore, the measurement of abnormal DNA methylation of the LRRC3B gene or the expression level of the LRRC3B gene of the present invention can be useful for the diagnosis of cancer, and the drug that regulates the abnormal methylation of the LRRC3B gene can be used as an anticancer agent by promoting the expression of the LRRC3B gene. Furthermore, since the LRRC3B gene or gene product of the present invention exhibits anticancer activity, an expression vector containing the LRRC3B gene and a transformant transformed with the expression vector may be used as an anticancer agent.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> LRRC3B <130> 4P-12-39 <160> 22 <170> KopatentIn 1.71 <210> 1 <211> 1718 <212> DNA <213> human LRRC3B cDNA gene <400> 1 gactgagccg actgcatctc tgggcaagcg gcgctttgcc tctctcctgt ctttgtctgc 60 tcttcctgca aggctacggc gcctggagca gggtctgcag cggccgccgc agcaacgcga 120 gccaagtcgt gccccgcacg gccgcccggg gccgcaccct cgctcggtgg cggcgcccga 180 agacgccagc cgcgccacgc actgcctgcg tcccgcgccc cagccgccgc gcaccaacgc 240 cgccgccttc gccgggagcc aagcccgccg ggccggcccg gtcccaggtg ggcagctcgg 300 cttgcaactg gttggaggtg gcggccgctg cagccctggc agcgggcaca cccctaagca 360 tacgcaccca acgcctcctc cctgagccac gaggatggag cagccacccc ggcccgggac 420 tggcgcaagg tgcccaagca aggaaagaaa taatgaagag acacatgtgt tagctgcagc 480 cttttgaaac acgcaagaag gaaatcaata gtgtggacag ggctggaacc tttaccacgc 540 ttgttggagt agatgaggaa tgggctcgtg attatgctga cattccagca tgaatctggt 600 agacctgtgg ttaacccgtt ccctctccat gtgtctcctc ctacaaagtt ttgttcttat 660 gatactgtgc tttcattctg ccagtatgtg tcccaagggc tgtctttgtt cttcctctgg 720 gggtttaaat gtcacctgta gcaatgcaaa tctcaaggaa atacctagag atcttcctcc 780 tgaaacagtc ttactgtatc tggactccaa tcagatcaca tctattccca atgaaatttt 840 taaggacctc catcaactga gagttctcaa cctgtccaaa aatggcattg agtttatcga 900 tgagcatgcc ttcaaaggag tagctgaaac cttgcagact ctggacttgt ccgacaatcg 960 gattcaaagt gtgcacaaaa atgccttcaa taacctgaag gccagggcca gaattgccaa 1020 caacccctgg cactgcgact gtactctaca gcaagttctg aggagcatgg cgtccaatca 1080 tgagacagcc cacaacgtga tctgtaaaac gtccgtgttg gatgaacatg ctggcagacc 1140 attcctcaat gctgccaacg acgctgacct ttgtaacctc cctaaaaaaa ctaccgatta 1200 tgccatgctg gtcaccatgt ttggctggtt cactatggtg atctcatatg tggtatatta 1260 tgtgaggcaa aatcaggagg atgcccggag acacctcgaa tacttgaaat ccctgccaag 1320 caggcagaag aaagcagatg aacctgatga tattagcact gtggtatagt gtccaaactg 1380 actgtcattg agaaagaaag aaagtagttt gcgattgcag tagaaataag tggtttactt 1440 ctcccatcca ttgtaaacat ttgaaacttt gtatttcagt ttcttttgaa ttatgccact 1500 gctgaacttt taacaaacac tacaacataa ataatttgag tttaggtgat ccacccctta 1560 attgtacccc cgatggtata tttttgagta agctactatt tgaacattag ttagatccat 1620 ctcactattt aataatgaaa tttatttttt taatttaaaa gcgaataaaa ggttaacttt 1680 gaaccatgga aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1718 <210> 2 <211> 259 <212> PRT <213> human LRRC3B protein <400> 2 Met Asn Leu Val Asp Leu Trp Leu Thr Arg Ser Leu Ser Met Cys Leu   1 5 10 15 Leu Leu Gln Ser Phe Val Leu Met Ile Leu Cys Phe His Ser Ala Ser              20 25 30 Met Cys Pro Lys Gly Cys Leu Cys Ser Ser Ser Gly Gly Leu Asn Val          35 40 45 Thr Cys Ser Asn Ala Asn Leu Lys Glu Ile Pro Arg Asp Leu Pro Pro      50 55 60 Glu Thr Val Leu Leu Tyr Leu Asp Ser Asn Gln Ile Thr Ser Ile Pro  65 70 75 80 Asn Glu Ile Phe Lys Asp Leu His Gln Leu Arg Val Leu Asn Leu Ser                  85 90 95 Lys Asn Gly Ile Glu Phe Ile Asp Glu His Ala Phe Lys Gly Val Ala             100 105 110 Glu Thr Leu Gln Thr Leu Asp Leu Ser Asp Asn Arg Ile Gln Ser Val         115 120 125 His Lys Asn Ala Phe Asn Asn Leu Lys Ala Arg Ala Arg Ile Ala Asn     130 135 140 Asn Pro Trp His Cys Asp Cys Thr Leu Gln Gln Val Leu Arg Ser Met 145 150 155 160 Ala Ser Asn His Glu Thr Ala His Asn Val Ile Cys Lys Thr Ser Val                 165 170 175 Leu Asp Glu His Ala Gly Arg Pro Phe Leu Asn Ala Ala Asn Asp Ala             180 185 190 Asp Leu Cys Asn Leu Pro Lys Lys Thr Thr Asp Tyr Ala Met Leu Val         195 200 205 Thr Met Phe Gly Trp Phe Thr Met Val Ile Ser Tyr Val Val Tyr Tyr     210 215 220 Val Arg Gln Asn Gln Glu Asp Ala Arg Arg His Leu Glu Tyr Leu Lys 225 230 235 240 Ser Leu Pro Ser Arg Gln Lys Lys Ala Asp Glu Pro Asp Asp Ile Ser                 245 250 255 Thr val val             <210> 3 <211> 807 <212> DNA <213> CpG island in exon 1 of human LRRC3B gene <400> 3 agagctgtgt aagggggtgc gaggaaggca ggcgagagca gcagagggag ttaatactcc 60 tacctcagct ggtctccggc agccaacgct cagcccccgc ctcctctctc cattccggac 120 cccgcaaccg cgaggcgaga gagcagcgcc ggcgaggcac tggctgggct ctgactgtca 180 cgttaattcc ctgctgcttt ttctagtccc cagccgcctc gcccctcctc cctcattccc 240 agcctgggtt tggaggcgat ttccctctat tgactgagcc gactgcatct ctgggcaagc 300 ggcgctttgc ctctctcctg tctttgtctg ctcttcctgc aaggctacgg cgcctggagc 360 agggtctgca gcggccgccg cagcaacgcg agccaagtcg tgccccgcac ggccgcccgg 420 ggccgcaccc tcgctcggtg gcggcgcccg aagacgccag ccgcgccacg cactgcctgc 480 gtcccgcgcc ccagccgccg cgcaccaacg ccgccgcctt cgccgggagc caagcccgcc 540 gggccggccc ggtcccaggt gggcagctcg gcttgcagct ggttggaggt ggcggccgct 600 gcagccctgg cagcgggcac acccctaagc atacgcaccc aacgcctcct ccctgagcca 660 cgaggatgga gcagccaccc cggcccggga ctggcgcaag gtaaggtgca gccgcctgtt 720 gcttttcgca gagcgcggga tcatccctct gcccgggtac ctaggccgtc cctccaagga 780 acaagtagag gagaactatt acaccag 807 <210> 4 <211> 19 <212> DNA <213> forward primer for CpG 72 cloning <400> 4 cgagagcagc agagggagt 19 <210> 5 <211> 19 <212> DNA <213> reverse primer for CpG 72 cloning <400> 5 gctctgcgaa aagcaacag 19 <210> 6 <211> 20 <212> DNA <213> forward primer of LRRC3B RT-PCR <400> 6 ttccctctcc atgtgtctcc 20 <210> 7 <211> 20 <212> DNA <213> reverse primer of LRRC3B RT-PCR <400> 7 ccagcatgtt catccaacac 20 <210> 8 <211> 21 <212> DNA <213> forward primer of beta-actin RT-PCR <400> 8 caagagatgg ccacggctgc t 21 <210> 9 <211> 21 <212> DNA <213> reverse primer of beta-actin RT-PCR <400> 9 tccttctgca tcctgtcggc a 21 <210> 10 <211> 19 <212> DNA <213> forward primer of CpG amplification for bisulfite sequencing <400> 10 tatttttagt ttgggtttg 19 <210> 11 <211> 25 <212> DNA <213> reverse primer of CpG amplification for bisulfite sequencing <400> 11 taattctcct ctacttattc cttaa 25 <210> 12 <211> 19 <212> DNA <213> forward primer of MSP # 1-M <400> 12 gcggtcgtcg tagtaacgc 19 <210> 13 <211> 21 <212> DNA <213> reverse primer of MSP # 1-M <400> 13 ccacctccaa ccaactacaa a 21 <210> 14 <211> 26 <212> DNA <213> forward primer of MSP # 1-U <400> 14 gtttgtagtg gttgttgtag taatgt 26 <210> 15 <211> 19 <212> DNA <213> reverse primer of MSP # 1-U <400> 15 ccaccacctc caaccaact 19 <210> 16 <211> 20 <212> DNA <213> forward primer of MSP # 2-M <400> 16 ggcgttcgaa gacgttagtc 20 <210> 17 <211> 20 <212> DNA <213> reverse primer of MSP # 2-M <400> 17 cgacccgacg aacttaactc 20 <210> 18 <211> 20 <212> DNA <213> forward primer of MSP # 2-U <400> 18 tgtttggtgg tggtgtttga 20 <210> 19 <211> 22 <212> DNA <213> reverse primer of MSP # 2-U <400> 19 ccaacccaac aaacttaact cc 22 <210> 20 <211> 20 <212> DNA <213> forward primer of LRRC3B-919 <400> 20 ccaagggctg tctttgttct 20 <210> 21 <211> 23 <212> DNA <213> reverse primer of LRRC3B-919A <400> 21 gagtctgcaa ggtttcagct act 23 <210> 22 <211> 23 <212> DNA <213> reverse primer of LRRC3B-919T <400> 22 gagtctgcaa ggtttcagct aca 23  

Claims (17)

LRRC3B protein set forth in SEQ ID NO: 2. The gene encoding the LRRC3B protein of claim 1. The gene of claim 2, wherein the gene is set forth in SEQ ID NO: 1. An expression vector comprising the LRRC3B gene of claim 2. A transformed cell line transformed with the expression vector of claim 4. Anticancer composition containing the LRRC3B gene of claim 2. An anticancer composition comprising the expression vector of claim 4. An anticancer composition comprising the transformed cell line of claim 5. An anticancer composition comprising a substance for controlling methylation of a nucleotide sequence including a CpG island near exon 1 of the LRRC3B gene described in SEQ ID NO: 3. 1) separating and purifying genomic DNA from the subject's tissue; 2) modifying the genomic DNA of step 1; 3) amplifying a portion of the nucleotide sequence of the CpG island of LRRC3B in the genomic DNA of step 2 by methylation-sensitive PCR (MSP); 4) detecting a methylation of the nucleotide sequence including the CpG island near exon 1 of the LRRC3B gene from the product of step 3. 1) separating and purifying the genomic DNA from the cancer tissue of the subject; 2) cleaving the genomic DNA of step 1 with a restriction enzyme that is sensitive to methylation of DNA and a restriction enzyme that is not sensitive to methylation; 3) performing PCR using a portion of the nucleotide sequence including the CpG island near exon 1 of the LRRC3B gene as a template using the DNA of step 2 as a primer; 4) detecting the methylation of the nucleotide sequence including the CpG island near exon 1 of the LRRC3B gene from the difference of the reaction product of step 3. 1) separating and purifying genomic DNA from the subject's tissue; 2) cleaving the genomic DNA of step 1 with a restriction enzyme sensitive to DNA methylation; 3) electrophoresis of the genomic DNA of step 2 followed by transfer to the membrane; 4) hybridizing the membrane of step 3 with a DNA probe labeled with radioisotopes on the CpG sequence of the LRRC3B gene; 5) A method for diagnosing cancer comprising exposing the membrane of step 4 to an X-ray film to detect methylation of a nucleotide sequence comprising a CpG island near exon 1 of the LRRC3B gene. 1) separating and purifying total RNA from the subject's tissue; 2) amplifying the expressed mRNA by RT-PCR using primers prepared with the entire RNA of step 1 as a partial sequence of the LRRC3B gene; 3) A cancer diagnostic method comprising the step of detecting the expression of LRRC3B gene from the amplified RNA of step 2. 1) separating and purifying total RNA from the subject's tissue; 2) transferring the entire RNA of step 1 to the membrane after electrophoresis; 3) hybridizing the LRRC3B gene labeled with the radioisotope to the membrane of step 2 with an RNA probe; 4) A method for diagnosing cancer comprising exposing the membrane of step 3 to an X-ray film to detect the expression level of the LRRC3B gene. The method according to any one of claims 10 to 14, wherein the tissue of step 1) is cancer tissue or blood tissue. Cancer diagnostic microchip comprising an antibody against LRRC3B protein. A method of diagnosing a disease using the SNP of the 919th base of the nucleotide sequence of SEQ ID NO: 1.

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